PROJECT SUMMARY The diagnosis and monitoring of patients with glioblastoma requires both surgery and magnetic resonance imaging in complement. Surgery critically provides tissue for histological and molecular characterization and MRI facilitates subsequent monitoring despite its limitation to differentiate between treatment response (radiation necrosis) and failure (tumor recurrence). As a result, patients undergo an additional surgical procedure to obtain tissue for molecular characterization, and to distinguish treatment response from failure requiring a change in therapy. Given the morbidity associated with surgery, patients cannot undergo serial biopsies therefore a minimally invasive test would provide real-time analysis and change our understanding and management of glioblastoma. Our laboratory is the first to identify both circulating tumor cells (CTCs) and exosomes (EVs) in the blood of patients with glioblastoma. In other cancers, CTCs and EVs have been independently captured, characterized, and analyzed to provide real-time information of the patient's tumor burden and identifying new mutations that confer resistance to therapy. Despite the blood brain barrier, EVs and CTCs are found in the blood in high and low frequencies. Currently, no existing technology exists to simultaneously capture and monitor both EVs and CTCs in patient blood. Thus, we propose the use of microfluidics to develop and validate our blood based `liquid biopsy' to isolate both EVs and CTCs from a single tube of blood from patients with glioblastoma at diagnosis and throughout treatment. We will also use standard techniques to evaluate ctDNA in parallel to evaluate any additive benefit to our assay. Microfluidic processing of clinical samples is low cost and shows great promise for translating `blood-on-a-chip' assays to the clinic. Our assay will allow for real-time molecular characterization throughout therapy and potentially identify novel treatments or better match patients with existing clinical trials. In addition, we aim to more accurately determine treatment response and inevitable recurrence using next generation sequencing of CTCs, EVs and ctDNA from diagnosis onward; ultimately establishing a minimally invasive test to diagnose, monitor, and detect recurrence in patients with glioblastoma. The new 2016 WHO classification of brain tumors has added molecular markers to the pathological diagnosis of glioblastoma which has traditionally required surgical tissue; we aim through our liquid biopsy to provide the first molecular characterization and classification of brain tumors through a simple blood test in real-time. This will dramatically advance our understanding and the care of patients with glioblastoma.